Deep sequencing reveals persistence of cell-associated mumps vaccine virus in chronic encephalitis

Chronic encephalitis with progressive loss of motor and cognitive function and high levels of intrathecal antibodies has been associated with persistent measles and rubella infections of the brain, most commonly in the context of subacute sclerosing pan-encephalitis (SSPE). We describe here a case of chronic panencephalitis in a child who had undergone successful allogeneic haematopoietic stem cell transplantation (allo-SCT) for severe combined immunodeficiency (SCID) in whom neither measles nor other pathogens could be detected. Using deep sequencing of fresh brain biopsy material, we identified the Jeryl Lynn 5 mumps virus (MuV^JL5), a component of the measles, mumps, rubella (MMR) vaccine that had been administered to the child before the diagnosis of SCID. Similar to findings in measles viruses recovered from cases of SSPE, the mumps virus genome from the brain showed evidence of biased hypermutation, particularly in the matrix (M) gene. Comparison with sequence data from the original vaccine batch used to immunise this child identified the expansion of variants present at low frequency in the vaccine as well as de novo fixed missense substitutions. This case represents the first conclusive demonstration of chronic panencephalitis due to mumps virus.

An 18-month-old male infant of consanguineous parents was diagnosed with SCID due to recombinase activating gene 1 (RAG1) deficiency (Fig. 1a) 4 months after MMR vaccination. The infant received a CD34-selected haploidentical allo-SCT. Full donor chimerism was rapidly achieved, with recovery of a diverse T cell repertoire (Fig. 1a) and excellent thymopoiesis. Post-transplant autoimmune cytopenias necessitated rituximab therapy, following which immunoglobulin replacement therapy was administered. Six months post-allo-SCT the child developed a febrile illness with rash, diarrhoea, lethargy and seizures, with evidence of encephalitis on magnetic resonance imaging (MRI) with raised CSF protein (1.35 g/L) and 11 lymphocytes. Despite extensive screening for neurotropic viruses and bacteria (Table S1), no pathogen was detected. He was treated with antimicrobials, antivirals and steroids, making a good recovery, and was discharged on anticonvulsant therapy. Over the next few months, the child was noted to have behavioural problems, hearing impairment and speech and language delay. One year after discharge, the seizures recurred with only partial response to antiepileptic treatment, but he remained stable for another 9 months. Repeat MRI scan 2 years after initial encephalitic illness showed no new lesions. Over the next few months, the child’s neurological condition deteriorated, with increasing seizures together with episodes of lethargy, disorientation, agitation, ataxic gait, visual loss and eventual hospitalisation.

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Repeat MRI of the brain at 40 months post-allo-SCT revealed abnormalities of grey and white matter, involving both cerebral hemispheres with multiple foci of contrast enhancement, including the basal ganglia, temporal and parieto-occipital cortices (Fig. 1b). In the absence of a firm diagnosis, a brain biopsy was taken.

Broad-spectrum antibiotics, aciclovir, ganciclovir and antifungal therapy were administered, as well as intravenous immunoglobulin (IVIG) and high-dose methyl prednisolone. Increasing seizures, left-sided weakness, cortical blindness and progressive global neurological deterioration over several weeks ended with the patient’s death 7 weeks following his last hospital admission.

CSF on last admission was acellular, but with oligoclonal bands, although total protein was low 0.12 g/L (normal 0.15–0.45 g/L. The oligoclonal bands were negative for HSV and VZV antibodies. Polyoma JC virus was detected by PCR in blood and urine on one occasion each (Table S2). CSF PCR was negative for 16S (bacterial) and 18S (fungal) rRNA and for a wide number of viral pathogens (Table S2), including JC virus, mumps, measles and rubella viruses. No pathogens were detected in stool, urine or blood (Table S2). CSF was negative for rubella and measles antibodies. In the absence of a firm diagnosis, a brain biopsy showed neuronal loss, astrocytic gliosis, reactive astrocytes, microglia and chronic inflammatory cells, but no viral inclusions or granulomata (Fig. 2a–e legend for detailed description). Infiltrating T lymphocytes were identified by CD3 staining (Fig. 2f).

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RNA-Seq of the brain biopsy [5, 20] resulted in 110 million 81 base pair (bp) paired-end reads. Approximately, one million reads were identified as non-human and were subsequently used for potential pathogen identification by the metaMix method [21]. Mumps virus was the only potential pathogen detected, with 77,624 assigned reads. The remaining reads were either unclassified or mapped to human and bacteria that are either environmental, part of human flora, kit contaminants [23] or of unknown significance (Table S3).

No reads mapped to measles, rubella or polyoma JC viruses. Using de novo assembly, the full-length viral sequence was recovered (99.94 % genome coverage, median coverage depth: 290) (Figure S1). Maximum likelihood phylogenetic analysis showed that the consensus viral sequence clustered closely with the MuV^JL5 vaccine strains (Figure S2) and shared 99.6 % identity with the publicly available sequence (GenBank: FJ211585) of the mumps component of the MMR preparation administered to the child. The sequence identified in this study is named MuV^JL5-London (GenBank: KX223397). PCR of RNA extracted from the brain biopsy confirmed the presence of MuV^JL5 vaccine strain (Table S2), but was negative for all other viruses, including polyoma JC (Table S2). Immunohistochemistry showed the presence of MuV nucleocapsid (N) protein with a neuronal pattern of staining (Fig. 2g, h). Control cortex and white matter were negative. We observed no specific staining for other pathogens (Fig. 2g,h for details) and no evidence of viral intracytoplasmic or intranuclear inclusion bodies. Retrospective testing showed weakly positive mumps antibody in the CSF.

To investigate this finding further, three million 251 bp paired-end reads were generated from the MMR vaccine batch that was used to immunise the child, of which 82,000 reads mapped to the MuV^JL5 vaccine strain (median coverage: 706, Figure S1). There were no fixed differences identified compared with the reference FJ211585. Fifteen positions in the vaccine sequence were found to be polymorphic (variant allele frequency (VAF) 5 % or greater, Table S4).

Of 55 fixed nucleotide differences between MuV^JL5-London and the vaccine, 12 had been present as a minority variant (10–24 %) in the vaccine (Figure S3, Table S4, VAF 75 % or greater). Of the remaining 43 new mutations, 28 coded for missense amino acid substitutions (Fig. 3, Table S5), with nine (32 %) located in the M protein, more than expected by chance (two-tailed exact binomial test p = 2.8e−04, Table S6). Other than position 140 in the M protein, the missense mutations were unique to MuV^JL5-London (Figure S6). The Tyr140His substitution in MuV^JL5-London is found in all wild-type strains as well as the more neurovirulent MuV^Ur Urabe and the MuV^JL2 Jeryl Lynn 2 vaccine strain, which is a minor component of some vaccines [1] (Figure S6).

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Prior to the introduction of routine MMR vaccination, mumps virus was the commonest cause of meningoencephalitis in children and was associated with serious sequelae including deafness and orchitis [11, 22]. No meningoencephalitis has been confirmed following the Jeryl lynn mumps vaccine, which this child received, and other adverse effects are rare [19] and usually self-limiting [14]. Mumps and rubella vaccine virus transcripts were detected in the brain of a child with IFNAR2 deficiency who developed fatal encephalitis following MMR vaccination [9], but brain immunohistochemistry was negative in this case (JB personal communication).

The presentation of this case, shares features with two previously published cases of MuV-related progressive encephalitis [13, 28]. Moreover, like measles SSPE and measles inclusion body encephalitis (MIBE) [29], which occurs in immunocompromised children within months of measles infection or vaccination, and unlike acute MuV encephalitis, no virus was detected in the CSF. However, other hallmarks of SSPE and MIBE such as high virus-specific antibody levels in CSF [29], were absent, although the child had deficient B cell function.

Importantly, however, in comparison with MuV^JL5 from the vaccine batch used to immunise the child, MuV^JL5-London showed evidence of T to C biased hypermutation, particularly in the M (matrix) gene suggestive of adenosine deaminase (ADAR)-driven mutation [10] (Table S4). The same finding in MV recovered from SSPE and MIBE [6, 29, 30] has been shown to reduce M protein expression [29] and is mooted to promote cell to cell spread and reduced viral assembly and shedding into the CSF. Biased hypermutation and lack of protein expression may also reflect the dispensability of M protein for neurovirulence [3].

MuV^JL5-London was characterised by the expansion of variants present at low frequency in the original vaccine, two of which have previously been described [8], as well as fixed de novo mutations in the M, N, P, L and F genes, a finding which again references those described for MV in SSPE. While most amino acid substitutions occurred in the M gene, in vitro data using the rat model implicates the F protein [3, 16] as key for mumps virus neurovirulence in acute encephalitis [16, 24] as well as for measles neurovirulence in SSPE [3]. The absence of within-host sequence diversity in the M and F ORFs (Figure S5, Table S7) further suggests functional constraints on these genes and supports directional selection acting on one or more of the amino acid changes in these proteins favouring spread and replication of MuV^JL5 in the brain.

In this child, clinical deterioration occurred following good T cell reconstitution in the context of normal T cell receptor repertoire and good levels of thymopoiesis. One possibility is that the presence of mumps tolerised and dysregulated T cell responses, thus permitting long-term viral persistence and the accumulation of pathogenic mutations. We made use of data on the donor’s HLA genotype to predict specific CTL cell epitopes (IEDB epitope prediction tool, percentile rank cutoff ≤1 %) in the MuV^JL5 mumps vaccine virus. Five missense mutations were located in the predicted CTL epitopes for the donor’s HLA (Fig. 3, Table S8). Of these, four had predicted T cell binding affinities at a level likely to correlate with T cell recognition [26] and reduced binding affinity in the mutated peptide (Table S8). This raises the possibility that partial escape of virus from immune surveillance through specific mutational changes may have occurred, especially as the same was not observed for peptides recognised by randomly selected HLA alleles (Supplementary Appendix, Table S8).

In summary, we report a case of progressive chronic encephalitis in a patient with SCID, in which the MuV^JL5 vaccine strain was detected in brain biopsy by deep sequencing. This case emphasises the generally poor rates of pathogen detection in encephalitides, making a strong case for deep sequencing of brain tissue where other methods have failed. The similar pattern of viral mutations in this case and those of measles SSPE suggest a common pathogenic process and further work is currently underway to determine the contribution of fixed mutations to the chronic encephalitic phenotype observed in the patient. It is important to note that MMR continues to be a highly effective and safe vaccine in the vast majority of individuals. However, this case highlights the importance of developing strategies such as newborn screening to exclude the very small proportion of infants at extremely high risk of complications from live-attenuated vaccines.